Human placental-derived allografts are biomaterials categorized as cellular, acellular, matrix-like products (CAMPs) that can serve as wound coverings due to placenta tissue's innate barrier function. The placental membrane consists of three layers, the amnion, the intermediate layer (IL), and the chorion, each contributing distinct functional and biological properties. This study investigates how variations in layer composition influence the Extracellular Matrix (ECM) and growth factor profiles of placental allografts. We compared Dual Layer (amnion-amnion), Full Thickness (amnion-intermediate-chorion, FT), and a novel four-layer allograft configuration (amnion-intermediate-chorion-amnion, ACA). Histological analyses using hematoxylin and eosin (H&E) and Masson's trichrome staining revealed distinct structural architecture among the three allografts, with FT and ACA exhibiting 4.9 times and 5.7 times greater thickness as compared with the Dual Layer, respectively. Compositional studies revealed different concentrations of key ECM components (collagen, elastin, proteoglycans, hyaluronic acid) and growth factors (ANG-2, EGF, PDGF-AA, VEGF) across allografts. The collagen concentration was two times higher in ACA as compared with the Dual Layer and FT. Additionally, FT and ACA demonstrated higher levels of growth factors and other ECM components, underscoring their biochemical diversity. These findings highlight the fact that the structural and biochemical properties of placental-derived allografts depend on their layer composition. This study underscores the importance of tailoring layer configurations that are optimized for clinical applications of CAMPs, enabling clinicians to select the most suitable grafts for clinical use, such as for wound management.